We worked on the development of new techniques that we (and others) can use in cell tracking/regenerative studies. ? (1) The green fluorescent protein (GFP) is among the most commonly used expression markers in biology. GFP-tagged cells have played a particularly important role in studies of cell lineage. Sensitive detection of GFP is crucially important for such studies to be successful, and problems with detection may account for discrepancies in the literature regarding the possible fate choices of stem cells. We developed a very sensitive technique for visualization of GFP. Using it, we can detect 90% of cells of donor origin while we could only see 50% of these cells when we employ the methods that are in general use in other laboratories. In addition, we showed that some cells permanently silence GFP expression. In the case of the progeny of bone marrow stem cells, it appears that the more distantly related they are to their precursors, the more likely it is that they will turn off the lineage marker (Toth, Shahar et al. 2007).? (2) After immunohistochemistry (IHC) began to be used routinely, a number of investigators worked on methods for staining multiple molecules in the same tissue sections or cells. Achieving this goal was not easy, however. One reason for this is that the majority of primary antibodies used in IHC reactions are raised in rabbits, and recognizing signals from two different rabbit antibodies is not trivial. Thus, all of the protocols described to date have serious limitations. We developed a simple, quick, and inexpensive solution to the problem. It has two major advantages over existing methods. First, by using antibodies from the same host, two or more antigens can be visualized in the same section with commercially available fluorescent dyes. Second, because the technique relies on signal amplification, both rare and abundant antigens can be detected (Toth and Mezey 2007).? (3) G proteincoupled receptors (GPCRs) mediate effects of extracellular signaling molecules in all the bodys cells. These receptors are encoded by scarce mRNAs; therefore, detecting their transcripts with conventional microarrays is difficult. We developed a method based on multiplex PCR and array detection of amplicons to assay GPCR gene expression with as little as 1 microgram of total RNA, and using it, we profiled three human bone marrow stromal cell (BMSC) lines (Hansen, Chen et al. 2007).? Using the histological techniques that we developed, we reported the differentiation of bone marrow derived CD45 (hematopoetic) expressing cells into uterine epithelium in the mice. The uterine endometrium is composed of epithelial and stromal cells, which undergo extensive degeneration and regeneration in every estrous cycle, and dramatic changes occur during pregnancy. The high turnover of cells requires a correspondingly high level of cell division by progenitor cells in the uterus but the character and source of these cells remain obscure. In our study, using a novel transgenic mouse, we showed that CD45 positive hematopoetic progenitor cells colonize the uterine epithelium, and that in pregnancy more than 80% of the epithelium can derive from these cells. Since we also found GFP positive uterine endothelial cells in long term GFP bone marrow transplanted mice, we concluded that circulating CD45+ cells play an important role in regenerating the uterine epithelium (Bratincsak, Brownstein et al. 2007). In another series of experiments we have demonstrated the contribution of bone marrow derived cells to salivary function in a mouse model of Sjogrens Syndrome (NOD mice). Applying a combination therapy of live splenocyte injections together with a stimulation through complete Freunds adjuvant (CFA) resulted in a complete restoration of salivary function in the NOD mice. This study establishes that a brief intervention in NOD mice with Sjogren's-like syndrome can reverse salivary gland dysfunction (Tran, Kodama et al. 2007).
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